PROJECT SUMMARY Atherosclerosis remains the leading cause of death and disability in the United States. Current therapeutic modalities for the treatment of severe atherosclerosis have high failure rates due to the development of restenosis secondary to neointimal hyperplasia. Nitric oxide (NO) is a small vasoprotective molecule that is known to inhibit the development of neointimal hyperplasia. As part of our prior VA Merit funding cycle, we developed several different NO-based therapies that successfully reduced neointimal hyperplasia in animal models. However, many veterans have diabetes mellitus (DM), a disease that alters the local arterial response to injury. In fact, patients with DM have been shown to have worse outcomes following vascular interventions due to aggressive neointimal hyperplasia. This led us to question whether NO-based therapies would be effective in this patient population. Preliminary data generated in our laboratory suggested that NO-based therapies have dramatically different efficacies in animal models of type I and type II diabetes. We found that NO was more efficacious at inhibiting neointimal hyperplasia in a rodent model of type II diabetes, yet completely ineffective in a rodent model of type I diabetes. These differences appear to be related to insulin levels. Thus, given that diabetes is common among veterans with severe atherosclerosis, and that these patients are most in need of a therapy to improve patency rates, it is critically important to further evaluate the efficacy of NO-based therapies in these diseases. Our hypothesis is that NO-mediated inhibition of neointimal hyperplasia is regulated by the insulin signaling pathway. Thus, the specific aims of this proposal are: Specific Aim 1: Characterize the effect of insulin and glucose on the ability of NO to regulate proliferation, migration, cell death, and reactive oxygen species in vascular smooth muscle cells (VSMC), endothelial cells, and adventitial fibroblasts in vitro. Specific Aim 2: Evaluate the efficacy of NO at inhibiting neointimal hyperplasia in vivo in animal models of type I and type II DM with and without insulin therapy. Specific Aim 3: Define the mechanism by which the insulin signaling pathway regulates the downstream beneficial effects of NO in the vasculature. Specifically, the mitogen activated protein kinase pathway will be manipulated using gain- and loss-of-function strategies. Preventing the development of neointimal hyperplasia in patients, especially those with diseases such as DM, will have a significant impact on patient care. Since patients with diabetes have an increased need for vascular interventions and have worse outcomes following interventions, this population would benefit greatly from a therapy that will improve outcomes. The studies in this proposal are novel, as no researcher has examined the role of NO in type I or type II DM. Thus, the data generated from this proposal will make a significant contribution to what is known about NO vascular biology and will lead to the development of innovative strategies to prevent neointimal hyperplasia in diabetic patient populations. Ultimately, by having a greater understanding of the pathways that regulate the downstream beneficial effects of NO in the vasculature, better NO-based therapies can be developed for our patients, thereby maximizing the beneficial potential this therapy will have for our veterans.